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McKenzie, Gregory J.; Craig, Nancy L.

doi:10.1186/1471-2180-6-39

Cited by 167 publications

(127 citation statements)

References 25 publications

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“…We ensured moderate translational expression of TetB by engineering a ribosome binding site (RBS) sequence that provided measurable resistance at minimal fitness cost (Materials and Methods; Salis et al , 2009). The final construct was integrated into the E. coli BW25113 chromosome using a site‐specific method for insertion at the Tn7 attachment sites downstream of the highly conserved glutamine synthetase gene, glmS (McKenzie & Craig, 2006). Our host strain BW25113 has a deletion in the genes necessary for arabinose catabolism; therefore, tet(B) expression is under constant induction in the presence of saturating arabinose, and we assume that P tot is constant within our system (Grenier et al , 2014).…”

Section: Resultsmentioning

confidence: 99%

“…Integration of tet(B) and variants into the chromosome of E. coli strain BW25113 [F‐, Δ(araD‐araB)567, ΔlacZ4787(::rrnB‐3), lambda‐, rph‐1, Δ(rhaD‐rhaB)568, hsdR514] was performed using a transposition‐based approach that uses Tn7 genes within pGRG36 to site‐specifically insert the pBAD/tet(B)/rrnB construct into the Tn7 attachment site downstream of the highly conserved glmS gene following the method described by McKenzie & Craig (2006). First, tet(B) WT and variant constructs were subcloned from pET28b(+) into pGRG36.…”

Section: Methodsmentioning

confidence: 99%

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Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer

Perez

Gomez

Kalvapalle

et al. 2017

Molecular Systems Biology

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The major facilitator superfamily (MFS) effluxers are prominent mediators of antimicrobial resistance. The biochemical characterization of MFS proteins is hindered by their complex membrane environment that makes in vitro biochemical analysis challenging. Since the physicochemical properties of proteins drive the fitness of an organism, we posed the question of whether we could reverse that relationship and derive meaningful biochemical parameters for a single protein simply from fitness changes it confers under varying strengths of selection. Here, we present a physiological model that uses cellular fitness as a proxy to predict the biochemical properties of the MFS tetracycline efflux pump, TetB, and a family of single amino acid variants. We determined two lumped biochemical parameters roughly describing K m and V max for TetB and variants. Including in vivo protein levels into our model allowed for more specified prediction of pump parameters relating to substrate binding affinity and pumping efficiency for TetB and variants. We further demonstrated the general utility of our model by solely using fitness to assay a library of tet(B) variants and estimate their biochemical properties.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer

Perez

Gomez

Kalvapalle

et al. 2017

Molecular Systems Biology

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“…A similar plasmid harboring EPEC espZ, pJSW101, was generated previously (18). To generate REPEC and EPEC espZ complements (VK581 and VK582, respectively), the REPEC ⌬espZ strain was transformed with pJSW201 or pJSW101 and screened for the Tn7-mediated transposition of espZ into the neutral site downstream of glmS (24).…”

Section: Methodsmentioning

confidence: 99%

The Secreted Effector Protein EspZ Is Essential for Virulence of Rabbit Enteropathogenic Escherichia coli

et al. 2015

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e Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (⌬espZ) and corresponding cis-complemented derivatives of rabbit enteropathogenic Escherichia coli and compared their abilities to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs, and intestinal tissue alterations. Consistent with data from previous reports, espZtransfected epithelial cells were refractory to infection-dependent effector translocation. Also, the ⌬espZ strain induced greater host cell death than did the parent and complemented strains. In rabbit infections, fecal ⌬espZ strain levels were 10-fold lower than those of the parent strain at 1 day postinfection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ⌬espZ mutant fecal carriage progressively decreased on subsequent days. ⌬espZ mutant-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of intestinal sections revealed increased epithelial cell apoptosis on day 1 after infection with the ⌬espZ strain compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host cell cytoprotection likely prevents epithelial cell death and sloughing and thereby promotes bacterial colonization.

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“…The resulting product was transformed into chemically competent E. coli TOP10 cells grown at 30°C. The transgene was inserted into the chromosomal Tn7 attachment site as described previously (71).…”

Section: Methodsmentioning

confidence: 99%

Stand-Alone EAL Domain Proteins Form a Distinct Subclass of EAL Proteins Involved in Regulation of Cell Motility and Biofilm Formation in Enterobacteria

et al. 2017

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The second messenger cyclic dimeric GMP (c-di-GMP) is almost ubiquitous among bacteria as are the c-di-GMP turnover proteins, which mediate the transition between motility and sessility. EAL domain proteins have been characterized as c-di-GMP-specific phosphodiesterases. While most EAL domain proteins contain additional, usually N-terminal, domains, there is a distinct family of proteins with stand-alone EAL domains, exemplified by Salmonella enterica serovar Typhimurium proteins STM3611 (YhjH/PdeH), a c-di-GMP-specific phosphodiesterase, and the enzymatically inactive STM1344 (YdiV/CdgR) and STM1697, which regulate bacterial motility through interaction with the flagellar master regulator, FlhDC. We have analyzed the phylogenetic distribution of EAL-only proteins and their potential functions. Genes encoding EAL-only proteins were found in various bacterial phyla, although most of them were seen in proteobacteria, particularly enterobacteria. Based on the conservation of the active site residues, nearly all stand-alone EAL domains encoded by genomes from phyla other than proteobacteria appear to represent functional phosphodiesterases. Within enterobacteria, EAL-only proteins were found to cluster either with YhjH or with one of the subfamilies of YdiV-related proteins. EAL-only proteins from Shigella flexneri, Klebsiella pneumoniae, and Yersinia enterocolitica were tested for their ability to regulate swimming and swarming motility and formation of the red, dry, and rough (rdar) biofilm morphotype. In these tests, YhjH-related proteins S4210, KPN_01159, KPN_03274, and YE4063 displayed properties typical of enzymatically active phosphodiesterases, whereas S1641 and YE1324 behaved like members of the YdiV/STM1697 subfamily, with Yersinia enterocolitica protein YE1324 shown to downregulate motility in its native host. Of two closely related EAL-only proteins, YE2225 is an active phosphodiesterase, while YE1324 appears to interact with FlhD. These results suggest that in FlhDC-harboring beta-and gammaproteobacteria, some EAL-only proteins evolved to become catalytically inactive and regulate motility and biofilm formation by interacting with FlhDC.IMPORTANCE The EAL domain superfamily consists mainly of proteins with cyclic dimeric GMP-specific phosphodiesterase activity, but individual domains have been classified in three classes according to their functions and conserved amino acid signatures. Proteins that consist solely of stand-alone EAL domains cannot rely on other domains to form catalytically active dimers, and most of them fall into one of two distinct classes: catalytically active phosphodiesterases with well-conserved residues of the active site and the dimerization loop, and catalytically inactive YdiV/ CdgR-like proteins that regulate bacterial motility by binding to the flagellar master

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Untitled

Cited by 167 publications

References 25 publications

Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer

Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer

The Secreted Effector Protein EspZ Is Essential for Virulence of Rabbit Enteropathogenic Escherichia coli

Stand-Alone EAL Domain Proteins Form a Distinct Subclass of EAL Proteins Involved in Regulation of Cell Motility and Biofilm Formation in Enterobacteria

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